On the Anticancer Effects of Cardiotonic Steroids

            Steroidal cardenolides and bufadienolides bind to the extracellular surface of Na⁺/K⁺-ATPase (the sodium/potassium pump), an integral membrane protein that translocates sodium and potassium ions across the cell membrane using ATP as the driving force. In addition to its transport function, it is less well known that Na⁺/K⁺-ATPase is also a versatile signaling protein whose aberrant expression is implicated in the development and progression of certain types of cancer. For example, in an in vitro cytotoxicity evaluation of a 3,187 compound library, Platz and co-workers identified digoxin (1) as a screening hit (mean IC₅₀ for inhibition of proliferation = 163 nM). Indeed, digoxin has long been used as a positive inotropic agent for the treatment of congestive heart failure and retrospective epidemiological studies of patients that received digoxin indicated that very few of them died from cancer. In the aforementioned study conducted by Platz et al, an analysis of the association between digoxin treatment and prostate cancer risk revealed that digoxin users had a 25% lower propensity for affliction with prostate cancer. Interestingly, the related steroid, 19-hydroxy-2’’-oxovoruscharine (2), isolated from milkweed plants, also displays potent anticancer activity against a range of cancer cell lines by means of potent binding to the Na⁺/K⁺-ATPase alpha1 subunit. This type of binding leads to deactivation of the cytoprotective effects caused by constitutively activated NF-kappaB, a phenomenon by which chemoresistant tumor cells typically evade cytotoxicity.

            Recently, the in vitro growth inhibitory properties of a relatively large series (>30) of bufadienolides (e.g. 3) were analyzed by MTT colorimetric assay (assay described here) in six human and two mouse cancer cell lines (J. Nat. Prod. 2013, In Press). Gamabufotalin rhamnoside (3) emerged as a cardiotonic steroid that displayed potent growth inhibitory effects when compared to digoxin (1) or ouabain. The observed antiproliferative activity of the bufadienolides tested in this series was markedly less pronounced in the mouse cancer cell lines studied. This can likely be attributed to two known mutations in the murine alpha1 subunit of Na⁺/K⁺-ATPase that reduce binding affinity for cardenolides and bufadienolides. In terms of structure-activity relationships, an alpa-oriented hydroxyl group at C11, in conjunction with a C14beta-OH, apparently contributed to growth inhibitory activity in human cancer cell lines. The latter is thought to donate a hydrogen bond to an asparagine in the Na⁺/K⁺-ATPase binding pocket. Finally, the pyrone unsaturated lactone moiety was shown to be essential for growth inhibition, as all of the active compounds in the series were functionalized with this heterocycle at carbon position 17. The analogous cardenolide congener of 3, containing a butenolide at C17 in place of the pyrone, is known as rhodexin A. Interestingly, rhodexin A exhibits very potent anticancer activity against human leukemia K562 cells (IC50 = 19 nM). The racemic total synthesis of rhodexin A is highlighted here.

            A number of potential mechanisms have been conjectured to account for cardiotonic steroid-induced growth inhibitory effects on cancer cells. One of those invokes inhibition of the glycolytic pathway culminating in reduced intracellular ATP levels. This is particularly detrimental to cancer cells, which have increased metabolic requirements for ATP. The precise mechanism by which a decrease in the activity of Na⁺/K⁺-ATPase produces glycolysis inhibition is not fully understood. The caveats of developing cardiotonic steroids as anticancer agents include potential cardiotoxic effects and a very narrow therapeutic index. Moreover, two oncology clinical trials involving digoxin failed to demonstrate significant clinical efficacy. Clinical evaluation of UNBS1450 is apparently ongoing in Europe. More fundamental research using specifically designed cardiotonic steroid derivatives is warranted in order to properly interrogate the notion of targeting the sodium/potassium pump as a novel treatment of malignancies.